The preliminary screening of 600 approved drugs against six phytopathogenic fungi at 100 μg ml−1 showed that 120, 162, 167, 85, 102 and 82 drugs against R. solani, S. sclerotiorum, B. cinerea, P. capsici, F. graminearum and F. oxysporum, respectively, inhibiting the growth of mycelium of by more than 70% (Fig. 1) . To further determine the antifungal activity of these drugs, they were evaluated using the EC50. We considered drugs with EC50 less than 25 μg ml−1 as candidates. Their original uses and toxicity are shown in Table 1 (https://pubchem.ncbi.nlm.nih.gov/, November 2022). As drug repurposing has gained tremendous popularity in the pharmaceutical field, we divided the candidate drugs into 9 lead series and conducted a brief discussion of structure and activity.

Fig. 1

A total of 600 drugs were tested against pathogenic fungi at 100 μg/mL

Table 1 Active drugs“hits”identified from initial screening anti-plant pathogenic fungiaFungicides against plant pathogenic fungi

Plant pathogens can cause crop yield reduction and quality deterioration, and control of plant diseases is still dominated by chemical fungicides. We evaluated the in vitro activity of the fungicide in Fig. 2. Carbendazim and thiophanate-methyl were broad-spectrum fungicides belonging to the benzimidazole and substituted benzene fungicides respectively, with EC50 values in the range of 0.14–22.12 μg ml−1 for pathogenic fungi. They had excellent activity against S. sclerotiorum, with EC50 was 0.68 and 0.53 μg ml−1, respectively. Difenoconazole is a sterol demethylation inhibitor with systemic, prophylactic and therapeutic effects. It had relatively potent activity against five pathogens except for R. solani, especially F. oxysporum, with an EC50 of 0.04 μg ml−1. Boscalid was a novel nicotinamide fungicide with positive action against R. solani, S. sclerotiorum, B. cinerea and F. graminearum, with EC50 < 2 μg ml−1. Azoxystrobin and kresoxim-methyl were strobilurin fungicides with good activity against S. sclerotiorum with EC50 of 4.9 and 4.66 μg ml−1, respectively. Pyrimethanil and thirluzamide belong to the genus of methyl pyrimidine and benzamides, respectively. They were potent pesticides against B. cinerea and R. solani with EC50 was 3.89 and 0.054 μg ml−1, respectively. We evaluated the different classes of fungicides against plant pathogens to provide a basis for the activity level of the drugs screened for this study.

Fig. 2

The EC50 of fungicides against phytopathogenic fungia. aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

Quinoline alkaloids

Alkaloids are a class of alkaline nitrogen-containing organic compounds in plants, marine organisms, microorganisms and insects. They have a wide range of biological activities such as lowering blood pressure, anti-tumor, central nervous system, lowering blood glucose, lowering blood lipids, insect repellent and anti-microbial [28,29,30,31,32]. Therefore they show potential for application in medical treatment and agricultural insecticide. In previous studies, our team designed and synthesized a variety of quinoline alkaloid derivatives based on different structures of natural product alkaloids, and tested their activity against plant pathogenic fungi [33,34,35,36,37,38,39,40] (Table 2), which provided a theoretical basis and laid a solid foundation for the development and application of alkaloids. To further obtain a broader spectrum of effective anti-phytopathogenic fungal alkaloids, the 28 drugs with different biological functions were repositioned (Table 3), and 6 compounds with better anti-phytopathogenic fungal activities were obtained, as shown in Table 1 and Fig. 3. Among them, pitavastatin calcium had a relatively broad spectrum of activity against pathogenic fungi, particularly against B. cinerea, P. capsici and F. oxysporum, with EC50 of less than 1 μg ml−1. However, cabozantinib showed more excellent activity against R. solani, with EC50 of 0.032 μg ml−1, which may be the introduction of 1-methoxy-4-methylbenzene into the quinoline structure to enhance the antifungal activity. In addition, dequalinium chloride, mefloquine hydrochloride and bedaquiline showed potential against B. cinerea or S. sclerotiorum. Therefore, the quinoline alkaloids designed and synthesized in our laboratory, as well as the repositioning of other functional alkaloids, we found that alkaloids have great potential in the field of agricultural disease control.

Table 2 The EC50 of quinoline alkaloids designed and synthesized in our laboratory against plant pathogenic fungiaTable 3 In vitro antifungal activities (inhibition rate/%) of the quinoline alkaloids against phytopathogenic fungiaFig. 3

The EC50 of quinoline alkaloids against phytopathogenic fungia. aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

Benzoimidazole/carbamate drugs

Benzimidazoles and their derivatives are an essential group of active agents in pesticides and pharmaceuticals with broad-spectrum biological activities, such as anticancer [41], antibacterial [42], antiviral [43] and antiparasitic [44]. Likewise, carbamates are a group of insecticides with outstanding bioactivity, which have properties such as rapid decomposition, short residual period and low bioaccumulation [45, 46]. On this basis, we screened 26 drugs against pathogenic fungi, as shown in Table 4, and screened out 11 drugs with excellent action, as shown in Table 1 and Fig. 4, which laid the foundation for searching for lead compounds with good activity.

Table 4 In vitro antifungal activities (inhibition rate/%) of the benzimidazole/carbamate drugs against phytopathogenic fungiaFig. 4

The EC50 of benzoimidazole/carbamate drugs against phytopathogenic fungia. aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

The structure-activity relationship showed that drugs attached to the benzene ring to n-butyl had positive activity against R. solani and S. sclerotiorum with EC50 of 0.051 μg ml−1 and 0.16 μg ml−1, respectively, while replacing the C atom in n-butyl with an S atom (fenbendazole) or O atom (oxibendazole) had an insignificant effect on activity. However, the S-atom in n-butyl was replaced by sulfur monoxide (albendazole S-oxide), significantly less active against both pathogens. The acetophenone structure (mebendazole) exhibited positive inhibition activity against R. solani and S. sclerotiorum. But the introduction of an F-atom into the acetophenone structure (flubendazole) significantly reduced the inhibition activity against R. solani (EC50 > 25 μg ml−1). Surprisingly, the substitution of the acetophenone with the phenyl sulfane moiety (fenbendazole) showed significant inhibitory activity against R. solani and S. sclerotiorum with EC50 of 0.007 μg ml−1 and 0.097 μg ml−1 respectively. However, the replacement of the S atom by the sulfoxide resulted in significantly reduced activity against both pathogens. By comparing the activity of benzimidazole/carbamate against plant pathogens, we found that iodopropynyl butylcarbamate was effective in expanding the antifungal spectrum and had promising activity against pathogenic fungi. Thus the repositioning of benzoimidazoles/carbamates can be an effective way to expand their application areas.

Azole drugs

Azoles drugs have a wide range of applications in agriculture and medicine, such as low cost, availability and bioavailability, making azoles drugs of choice treating of fungal infections in most HIV/AIDS patients [47]. In agricultural production, triazole fungicides are mainly used to control plant fungal diseases caused by rust and mulberry powdery mildew pathogens due to their high efficiency and low toxicity [48]. The results indicate that azoles have broad antifungal activity as an essential backbone, which offers the possibility of developing new drugs. We screened 46 azole drugs (Table 5) against plant pathogens and obtained 16 drugs with optimal activity, as shown in Table 1 and Fig. 5.

Table 5 In vitro antifungal activities (inhibition rate/%) of the azole drugs against phytopathogenic fungiaFig. 5

The EC50 of azole drugs against phytopathogenic fungia. aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

The activity of bifonazole and clotrimazole showed that clotrimazole was more active than bifonazole against R. solani and S. sclerotiorum, which may be related to 1-benzyl-1H-imidazole. Econazole, vagistat, isoconazole nitrate and fenticonazole nitrate shared the basic structure (1-(2-(2,4-dichlorophenyl)-methoxy-2-ethyl)−1H-imidazole) and had comparable activity against all pathogens. All the compounds showed excellent activity against P. capsici with EC50 < 0.06 μg ml−1, indicating that this basic structure played a vital role in anti-pathogenic fungi. Replacing the O atom in the basic structure above with an S atom (sulconazle nitrate) had little effect on the activity against the plant pathogens, suggesting that the basic structure was still the key to activity. Voriconazole, efinaconazole and isavuconazole had similar basic structures, but efinaconazole showed better activity than the other two drugs with EC50 of 0.095 μg ml−1 and 0.035 μg ml−1 against S. sclerotiorum and F. oxysporum, respectively. The activity of ketoconazole against plant pathogens was significantly higher than that of terconazole, and the EC50 was in the range of 0.12–2.34 μg ml−1, which showed that 1-methyl-1H-imidazole was more effective than 1-methyl-1H − 1,2,4-triazole in this type of drug. However, not all drugs containing 1-methyl-1H − 1,2,4-triazole structures were less active against pathogens than 1-methyl-1H-imidazole. Itraconazole and posaconazole showed the strongest inhibitory activity against pathogens with EC50 < 0.17 μg ml−1. In summary, the azole backbone is the main active group against plant pathogenic fungi with a view to repositioning old drugs for plant disease control.

Isothiazolinone drugs

Isothiazolinone is a major industrial bactericide, antiseptic and anti-enzyme agent, with outstanding inhibition of mold, algae and other microorganisms [49]. Recently, a series of derivatives with anti-tuberculosis and lipase inhibitors have been designed and synthesized [50, 51]. We selected 26 isothiazolinones (Table 6) for screening against phytopathogenic fungi and obtained 8 drugs with good activity, which were briefly analysed in Table 1 and Fig. 6.

Table 6 In vitro antifungal activities (inhibition rate/%) of the isothiazolinone drugs against phytopathogenic fungiaFig. 6

The EC50 of isothiazolinone drugs against phytopathogenic fungia. aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

The 5-chloro-3-hydroxyisothiazole was the introduction of a Cl atom to the isothiazol-3-one structure, which significantly increased the activity against plant fungi with an EC50 in the range of 0.98–4.06 μg ml−1, but the introduction of a methyl group to 5-chloro-3-hydroxyisothiazole decreased the antifungal activity. The introduction of a Cl atom and octane on the 5-chloro-3-hydroxyisothiazole structure resulted in increasing activity against phytopathogenic fungi with an EC50 in the range of 0.27–2.64 μg ml−1. However, 2-octyl-2H-isothiazol-3-one showed comparable activity against plant pathogens compared to 4,5-dichloro-2-octyl-isothiazolone. Therefore, the introduction of octane in this structure may enhance the activity of phytopathogenic fungi, compared to 1,2-benzisothiazol-3(2H)-one, 2-methyl-1,2-benzothiazol-3(2H)-one and 6-fluoro-1,2-benzoisothiazol-3(2H)-one showed reduced antifungal activity, indicating that the introduction of substituents in this structure (benzoisothiazole) reduced the antifungal activity. Overall, the isothiazolinone structure is a potential lead compound against phytopathogenic fungi.

Pyrimidine drugs

Pyrimidine derivatives play an important role in insecticide, fungicide, weed control, antiviral, anticancer, etc. [52, 53], and have been the focus of attention of major pesticide companies in the world. In this study, we screened 65 drugs (Table 7) against agropathogenic fungi and obtained 10 highly active drugs, as shown in Table 1 and Fig. 7.

Table 7 In vitro antifungal activities (inhibition rate/%) of the pyrimidine drugs against phytopathogenic fungiaFig. 7

The EC50 of pyrimidine drugs against phytopathogenic fungia. aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

Taking 5-fluorouracil as a backbone, a molecule ((2R,3S,4R,5S)−2-(hydroxymethyl)-tetrahydrofuran-3,4-diol) was introduced to become 5-fluorouridine, which significantly enhanced its activity against plant pathogenic fungi. Compared with 5-fluorouridine, the structure of floxuridine was one less OH group, but it was slightly less active against S. sclerotiorum and B. cinerea. It showed that the introduction of this moiety directly affected the anti-pathogenic fungal activity of the compound. Compared to ganciclovir, 2'-deoxyguanosine was less active against S. sclerotiorum. The pyrimidine-4-amine-based compounds showed inhibitory activity against B. cinerea with an EC50 range of 2.29–13.52 μg ml−1. Both dabrafenib and sulfatinib contain N-methylmethanesulfonamide, which were active against S. sclerotiorum and B. cinerea, and had superior antifungal activity to sulfamitinib. Thus, the activity of pyrimidine analogues against phytopathogenic fungi are based on the pyrimidine structure with other moieties, which are beneficial to improve the activity and can be used as candidate lead compounds against plant pathogenic fungi.

Pyridine drugs

In agriculture, pyridines are used as insecticides, herbicides and plant growth regulators. In particular, in herbicides, a number of highly effective and low-toxicity varieties have been developed, such as pyrimethanesulfuron, pirimicarb and acetamiprid [54]. In this study, 31 drugs that have not yet been applied against plant pathogenic fungi were screened, as shown in Table 8, and 12 drugs with application potential were finally screened out, as shown in Table 1. We aim to obtain lead structures or drugs with triple action of insecticide, herbicide, and disease control.

Table 8 In vitro antifungal activities (inhibition rate/%) of the pyridines drugs against phytopathogenic fungia

As shown in Fig. 8, nilvadipine, nimodipine, amlodipine and amlodipine maleate belonged to the dihydropyridine group and showed activity against B. cinerea, among which nilvadipine had the strongest activity with an EC50 of 5.74 μg ml−1. This may be related to the electron-absorbing groups attached to the pyridine ring. Amlodipine maleate was a salt form of amlodipine with a slightly increased activity against B. cinerea. Liranaftate and pyributicarb had broad-spectrum and excellent activity against plant phytopathogens. Compared with liranaftate and pyributicarb, the activity of benzene ring-linked the cyclohexane ring with benzene ring-linked tert-butyl ring was one order of magnitude higher against five pathogenic fungi except F. oxysporum, among which the activity against B. cinerea was the best, with EC50 of 0.004 μg ml−1. The results suggest that pyridines, especially liranaftate and pyributicarb are promising for repositioning as fungicides for the control of plant pathogens.

Fig. 8

The EC50 of pyridine drugs compounds against phytopathogenic fungia. aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

Piperidine/Piperazine drugs

Piperidine ring and piperazine group are often introduced into many drug molecules to improve the pharmacokinetic properties by effectively adjusting the ratio of lipid-water distribution and acid-base balance of drugs, which improves the bioavailability of drug molecules and drug efficacy [55,56,57,58]. In this study, mainly 65 antipsychotics were used to screen agricultural fungi, as shown in Table 9, and 18 drugs with relatively good activity were obtained as shown in Table 1.

Table 9 In vitro antifungal activities (inhibition rate/%) of the piperidine/piperazine drugs against phytopathogenic fungia

As shown in Fig. 9, the drugs with piperazine and piperidine structures include two forms of N-methyl group on the outside and inside, and the two forms of piperazine drugs have little difference against antifungal activity. But loratadine and penfluridol had excellent activity with EC50 of 6.19 μg ml−1 and 6.59 μg ml−1 against R. solani and S. sclerotiorum, respectively. Compared with the piperazine structure with the N-methyl position on the outside, the piperidine structure had better anti-phytopathogenic activity. Among them, trifluoperazine not only had significant antifungal activity but also expanded the antifungal spectrum. In addition, ponatinib had the best activity against R. solani. The EC50 was 0.017 μg ml−1. Therefore, piperazine and piperidine compounds have the potential to develop drugs against agricultural pathogenic fungi.

Fig. 9

The EC50 of piperidine/piperazine drugs against phytopathogenic fungia. aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

Ionic liquids

Ionic liquids are considered a friendly solvent and commonly used in the extraction of natural products. They are mainly classified as quaternary ammonium ionic liquids, pyridine ionic liquids, quaternary phosphate ionic liquids and imidazole ionic liquids [59, 60]. This study used 37 ionic liquids to inhibit plant pathogens as shown in Table 10. According to Table 1 and Fig. 10, 15 potential drugs were briefly analyzed in order to apply them to agriculture.

Table 10 In vitro antifungal activities (inhibition rate/%) of the ionic liquids against phytopathogenic fungiaFig. 10

The EC50 of ionic liquids against phytopathogenic fungia. aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

In all ionic liquids, we found that the longer the carbon chain of the drug, the better the activity against S. sclerotiorum. 1-Dodecyl-3-methylimidazolium chloride products better active with EC50 of 6.12 μg ml−1 against S. sclerotiorum. Compared to 1-dodecylpyridinium bromide, 1-tetradecylpyridinium chloride was less active against phytopathogenic fungi despite the carbon-chain length, so the effect on antifungal activity may be ion-related. 1-Dodecylpyridinium with the bromine ion increased the activity against S. sclerotiorum, B. cinerea and F. oxysporum. Compared to myristalkonium chloride, the carbon chain increased and the activity was enhanced against S. sclerotiorum with cetalkonium chloride EC50 of 8.36 μg ml−1, but significantly decreased activity against B. cinerea. Compared with benzyldodecyldimethylammonium bromide, chloride ion replaced by bromine ion dodecyl dimethyl benzyl ammonium bromide increased the activity of S. sclerotiorum and B. cinerea. The EC50 values were 5.80 μg ml−1 and 8.85 μg ml−1, respectively. In summary, the carbon chain length of the ionic liquid drugs had a significant effect on the resistance to phytopathogenic fungi. Compared to chlorohexidine diacetate, enebicyanog had a narrower spectrum of activity against phytopathogenic fungi, but it had better activity against S. sclerotiorum and B. cinerea, with EC50 of 0.91 μg ml−1 and 0.62 μg ml−1 respectively. Therefore, ionic liquids are expected to be used in the control of plant resistant pathogenic fungi.

Miscellaneous group drugs

Miscellaneous group drugs against plant pathogenic fungi are shown in Table 11 and Table 1. Some drugs with relatively broad anti-pathogenic activity were selected for a brief analysis as shown in Fig. 11.

Table 11 In vitro antifungal activities (inhibition rate/%) of the miscellaneous group against phytopathogenic fungiaFig. 11

The EC50 of miscellaneous drugs against phytopathogenic fungia. aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

Monensin, natamycin and griseofulvin are antibiotics, but they have different effects, which monensin inhibits the growth of coccidia, gram-positive bacteria, algae and protozoa [61]. Natamycin is commonly used as a preservative to prevent mould contamination in food [62, 63]. Griseofulvin is widely used in clinical medicine to treat skin and stratum corneum fungal infections, and also in the prevention and treatment of fungal diseases in agriculture [64]. Monensin sodium salt, natamycin and griseofulvin had broad-spectrum activity against plant pathogenic fungi, with EC50 ranging from 0.076 to 13.20 μg ml−1. Butenafine hydrochloride, terbinafine hydrochloride and tolnaftate are a group of antifungal drugs, which are applied to the treatment of tinea capitis and other tinea diseases [65, 66]. In this screening, butenafine hydrochloride, terbinafine hydrochloride and tolnaftate were also found to have excellent activity against pathogenic fungi, with EC50 in the range of 0.07–18.05 μg ml−1. It was worth noting that they had significant activity in B. cinerea, with EC50 of 0.07, 0.11 and 0.07 μg ml−1, respectively. Oxyclozanide is the drug of choice for clinical anti-helminth infections, which has the characteristics of broad spectrum, low toxicity and low residue [67]. Through drug repositioning strategy, we found that oxyclozanide also had excellent activity against phytopathogenic fungi with EC50 in the range of 0.09–0.71 μg ml−1. Carbonyl cyanide 3-chloro-phenylhydrazone (CCCP) is an inhibitor of oxidative phosphorylation that disrupts the mitochondrial membrane potential [68]. The evaluation of the in vitro activity of CCCP against pathogenic fungi revealed a broad antifungal spectrum and potent activity with EC50 in the range of 0.38–6.07 μg ml−1. Although this group of drugs was not analyzed by activity and structure, these results provided a structure-based screening approach to repurpose commercially available drugs with the expectation of discovering broad-spectrum, effective drugs against plant pathogens.